The present invention relates to a process for accumulating α-keto butyrate. The present invention also relates to the use of a natural mutant micro-organism and/or extracted enzyme to produce natural α-keto butyrate. The present invention also relates to a process of obtaining emoxyfurone.
Alpha-keto butyrate, a synonym of α-keto-butyric acid, 2-oxo-butyrate, 2-oxo-butyric acid, was accumulated by various organisms. According to contradictory reports, however, the molecule was also shown to be produced in balanced concentration ranges and, therefore, not to be suitable for accumulation.
In JP 03183476, it is shown that a micro-organism belonging to the genus Pseudomonas is capable of converting 2,3-dihydroxy butyric acid into α-keto butyric acid. However, Pseudomonas is not a food-grade micro-organism and, therefore, the α-keto butyric acid obtained by fermentation of this micro-organism is not easily applicable to food products. This is also valid for derivatives obtained by further processing of the α-keto butyric acid obtained by this process.
Nakahara, Nkajima-Kambe and Sato (Biotechnol. Lett. (1994), 16(3), 263–8) have shown that α-keto butyric acid can be accumulated by a Rhodococcus equi strain. However, in this bioconversion, 1,2-propanediol has to be added to the medium, which is not preferred for obtaining food ingredients. Additionally, it is not warranted that the micro-organism is harmless for producing food ingredients or reactants in the synthesis of food ingredients.
There are further reports of accumulation of α-keto butyric acid in recombinant micro-organisms (Kisumi, Sugiura, Takagi and Chibata, J. Antibiot. (Tokyo) 1977 Jan. 30(1), 111–7). However, for the further synthesis of flavors (food-ingredients) it is wished to get α-keto butyric acid from genetically not modified organisms.
Sluis et al (Sluis, Wolken, Giuseppin, Tramper Wijffels, “Effect of threonine, cysthionine, and the branched-chain amino acids on the metabolism of Zygosaccharomyces rouxi”, Enzyme Microb Technol, 2000 Feb. 1; 26(2–4): 292–300) found no accumulation of α-keto-butyrate in a fungal strain, because the α-keto-butyrate pool size in this micro-organism was in balance all the time.
It was already shown that high levels of α-keto-butyrate may be toxic to a micro-organism (Fisher, Eisenstein, “An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from E. coli”, J Bacteriol, 1993 October; 175(20): 6605–13). In conclusion, accumulation of α-keto-butyrate may be difficult to achieve by bioconversion.
In the light of the prior art, it is an objective to obtain α-keto butyric acid in a biotechnological process. In particular, it is an objective to obtain α-keto butyric acid from a natural mutant in contrast to a genetically engineered micro-organism. In addition, a micro-organism suitable for the accumulation or production of α-keto butyric acid should be a food-grade micro-organism, in order to warrant an unobjectionable and harmless use in food-products.
In particular, α-keto butyrate is the starting point for obtaining specific furanones, for example the “Maggi-lactone” (5-ethyl-3-hydroxy-4-methy-2(5H)-furanone), which has a very strong but specific flavor and which is used in small concentrations as a “top-note”. It thus would be desirable to find an alternative to the chemically synthesized α-keto butyrate that is so far used as an intermediate for further synthesis of flavors.